Study of retinoic acid signaling in cancer cells: Cloning and characterization of retinoic acid responsive genes

Retinoids such as all-trans-retinoic acid (ATRA) are promising agents for cancer chemoprevention and therapy. ATRA can cause growth inhibition, induction of differentiation and apoptosis of a variety of cancer cells. These effects are thought to be mediated by nuclear retinoids receptors which are involved in ligand-dependent transcriptional activation of downstream target genes. Using differential display, we identified several retinoic acid responsive genes in the head and neck squamous carcinoma cells and lung cancer cells, including tissue type transglutaminase, cytochrome P450-related retinoic acid hydroxylase, and a novel gene, designated RAIG1. RAIG1 has two transcripts of 2.4 and 6.8 kbp, respectively, that are generated by alternative selection of polyadenylation sites. Both transcripts have the same open reading frame that encodes a protein comprised of 357 amino acid residues. The deduced RAIG1 protein sequence contains seven transmembrane domains, a signature structure of G protein-coupled receptors. RAIG1 mRNA is expressed at high level in fetal and adult lung tissues. Induction of RAIG1 expression by ATRA is rapid and dose-dependent. A fusion protein of RAIG1 and the green fluorescent protein was localized in the cell surface membrane and perinuclear vesicles in transiently transfected cells. The locus for RAIG1 gene was mapped to a region between D12S358 and D12S847 on chromosome 12p12.3-p13. Our study of the novel retinoic acid induced gene RAIG1 provide evidence for a possible interaction between retinoid and G protein signaling pathways.^ We further examined RAIG1 expression pattern in a panel of 84 cancer cell lines of different origin. The expression level varies greatly from very high to non-detectable. We selected a panel of different cancer cells to study the effects of retinoids and other differentiation agents. We observed: (1) In most cases, retinoids (including all-trans retinoic acid, 4HPR, CD437) could induce the expression of RAIG-1 in cells from cancers of the breast, colon, head and neck, lung, ovarian and prostate. (2) Compare to retinoids, butyrate is often a more potent inducer of RAIG-1 expression in many cancer cells. (3) Butyrate, Phenylacetate butyrate, (R)P-Butyrate and (S)P-Butyrate have different impact on RAIG1 expression which varies among different cell lines. Our results indicate that retinoids could restore RAIG1 expression that is down-regulated in many cancer cells.^ A mouse homologous gene, mRAIG1, was cloned by 5$\sp\prime$ RACE reaction. mRAIG1 cDNA has 2105 bp and shares 63% identity with RAIG1 cDNA. mRAIG1 encodes a polypeptide of 356 amino acid which is 76% identity with RAIG1 protein. mRAIG1 protein also has seven transmembrane domains which are structurally identical to those of RAIG1 protein. Only one 2.2 kbp mRAIG1 transcript could be detected. The mRAIG1 mRNA is also highly expressed in lung tissue. The expression of mRAIG1 gene could be induced by ATRA in several mouse embryonal carcinoma cells. The induction of mRAIG1 expression is associated with retinoic acid-induced neuroectoderm differentiation of P19 cells. Similarity in cDNA and protein sequence, secondary structure, tissue distribution and inducible expression by retinoic acid strongly suggest that the mouse gene is the homologue of the human RAIG1 gene. ^

Studies of subcellular localization and complex formation of the Agrobacterium tumefaciens transport ATPase VirB11

The VirB11 ATPase is an essential component of an Agrobacterium tumefaciens type IV bacterial secretion system that transfers oncogenic nucleoprotein complexes to susceptible plant cells. This dissertation investigates the subcellular localization and homo-oligomeric state of the VirB11 ATPase in order to provide insights about the assembly of the protein as a subunit of this membrane-associated transfer system. Subcellular fractionation studies and quantitative immunoblot analysis demonstrated that $\sim$30% of VirB11 partitioned as soluble protein and $\sim$70% was tightly associated with the bacterial cytoplasmic membrane. No differences were detected in VirB11 subcellular localization and membrane association in the presence or absence of other transport system components. Mutations in virB11 affecting protein function were mapped near the amino terminus, just upstream of a region encoding a Walker 'A' nucleotide-binding site, and within the Walker 'A' motif partitioned almost exclusively with the cytoplasmic membrane, suggesting that an activity associated with nucleotide binding could modulate the affinity of VirB11 for the cytoplasmic membrane. Merodiploid analysis of VirB11 mutant and truncation derivatives provided strong evidence that VirB11 functions as a homo- or heteromultimer and that the C-terminal half of VirB11 contains a protein interaction domain. A combination of biochemical and molecular genetic approaches suggested that VirB11 and the green fluorescence protein (GFP) formed a mixed multimer as demonstrated by immunoprecipitation experiments with anti-GFP antibodies. Second, a hybrid protein composed of VirB11 fused to the N-terminal DNA-binding domain of bacteriophage $\lambda$ cI repressor conferred immunity to $\lambda$ superinfection, demonstrating that VirB11 self-association promotes dimerization of the chimeric repressor. A conserved Walker 'A' motif, though required for VirB11 function in T-complex export, was not necessary for VirB11 self-association. Sequences in both the N- and the C-terminal halves of the protein were found to contribute to self-association of the full length protein. Chemical cross-linking experiments with His$\sb6$ tagged VirB11 suggested that VirB11 probably assembles into a higher order homo-oligomeric complex. ^

The AtProT family. Compatible solute transporters with similar substrate specificity but differential expression patterns

Proline transporters (ProTs) mediate transport of the compatible solutes Pro, glycine betaine, and the stress-induced compound gamma-aminobutyric acid. A new member of this gene family, AtProT3, was isolated from Arabidopsis (Arabidopsis thaliana), and its properties were compared to AtProT1 and AtProT2. Transient expression of fusions of AtProT and the green fluorescent protein in tobacco (Nicotiana tabacum) protoplasts revealed that all three AtProTs were localized at the plasma membrane. Expression in a yeast (Saccharomyces cerevisiae) mutant demonstrated that the affinity of all three AtProTs was highest for glycine betaine (K-m = 0.1-0.3 mM), lower for Pro (K-m = 0.4-1 mM), and lowest for gamma-aminobutyric acid (K-m = 4-5 mM). Relative quantification of the mRNA level using real-time PCR and analyses of transgenic plants expressing the beta-glucuronidase (uidA) gene under control of individual AtProT promoters showed that the expression pattern of AtProTs are complementary. AtProT1 expression was found in the phloem or phloem parenchyma cells throughout the whole plant, indicative of a role in long-distance transport of compatible solutes. beta-Glucuronidase activity under the control of the AtProT2 promoter was restricted to the epidermis and the cortex cells in roots, whereas in leaves, staining could be demonstrated only after wounding. In contrast, AtProT3 expression was restricted to the above-ground parts of the plant and could be localized to the epidermal cells in leaves. These results showed that, although intracellular localization, substrate specificity, and affinity are very similar, the transporters fulfill different roles in planta.

Interaction of Plectin with Keratins 5 and 14: Dependence on Several Plectin Domains and Keratin Quaternary Structure.

Plectin, a cytolinker of the plakin family, anchors the intermediate filament (IF) network formed by keratins 5 and 14 (K5/K14) to hemidesmosomes, junctional adhesion complexes in basal keratinocytes. Genetic alterations of these proteins cause epidermolysis bullosa simplex (EBS) characterized by disturbed cytoarchitecture and cell fragility. The mechanisms through which mutations located after the documented plectin IF-binding site, composed of the plakin-repeat domain (PRD) B5 and the linker, as well as mutations in K5 or K14, lead to EBS remain unclear. We investigated the interaction of plectin C terminus, encompassing four domains, the PRD B5, the linker, the PRD C, and the C extremity, with K5/K14 using different approaches, including a rapid and sensitive fluorescent protein-binding assay, based on enhanced green fluorescent protein-tagged proteins (FluoBACE). Our results demonstrate that all four plectin C-terminal domains contribute to its association with K5/K14 and act synergistically to ensure efficient IF binding. The plectin C terminus predominantly interacted with the K5/K14 coil 1 domain and bound more extensively to K5/K14 filaments compared with monomeric keratins or IF assembly intermediates. These findings indicate a multimodular association of plectin with K5/K14 filaments and give insights into the molecular basis of EBS associated with pathogenic mutations in plectin, K5, or K14 genes.Journal of Investigative Dermatology advance online publication, 10 July 2014; doi:10.1038/jid.2014.255.

Autophagy in Trypanosoma brucei: amino acid requirement and regulation during different growth phases.

Autophagy in the protozoan parasite, Trypanosoma brucei, may be involved in differentiation between different life cycle forms and during growth in culture. We have generated multiple parasite cell lines stably expressing green fluorescent protein- or hemagglutinin-tagged forms of the autophagy marker proteins, TbAtg8.1 and TbAtg8.2, in T. brucei procyclic forms to establish a trypanosome system for quick and reliable determination of autophagy under different culture conditions using flow cytometry. We found that starvation-induced autophagy in T. brucei can be inhibited by addition of a single amino acid, histidine, to the incubation buffer. In addition, we show that autophagy is induced when parasites enter stationary growth phase in culture and that their capacity to undergo starvation-induced autophagy decreases with increasing cell density.

Development of small molecular tools for the cellular study of adenosine receptors

The adenosine receptors are members of the G-protein coupled receptor (GPCR) family which represents the largest class of cell-surface proteins mediating cellular communication. As a result, GPCRs are formidable drug targets and it is estimated that approximately 30% of the marketed drugs act through members of this receptor class. There are four known subtypes of adenosine receptors: A1, A2A, A2B and A3. The adenosine A1 receptor, which is the subject of this presentation, mediates the physiological effects of adenosine in various tissues including the brain, heart, kidney and adipocytes. In the brain for instance, its role in epilepsy and ischemia has been the focus of many studies. Previous attempts to study the biosynthesis, trafficking and agonist-induced internalisation of the adenosine A1 receptor in neurons using fluorescent protein-receptor fusion constructs have been hampered by the sheer size of the fluorescent protein (GFP) that ultimately affected the function of the receptor. We have therefore initiated a research programme to develop small molecule fluorescent agonists that selectively activate the adenosine A1 receptor. Our probe design is based on the endogenous ligand adenosine and the known unselective adenosine receptor agonist NECA. We have synthesised a small library of non-fluorescent adenosine derivatives that have different cyclic and bicyclic moieties at the 6 position of the purine ring and have evaluated the pharmacology of these compounds using a yeast-based assay. This analysis revealed compounds with interesting behaviour, i.e. exhibiting subtype-selectivity and biased signalling, that can be potentially used as tool compounds in their own right for cellular studies of the adenosine A1 receptor. Furthermore, we have also linked fluorescent dyes to the purine ring and discovered fluorescent compounds that can activate the adenosine A1 receptor.

Successful transnasal delivery of stem cells in a rat model of perinatal hypoxic-ischemic brain injury

OBJECTIVE: New routes for cell transplantation into the brain need to be explored as intracerebral or intrathecal applications have a high risk to cause damage to the central nervous system. It has been hypothesized that transnasally administrated cells bypass the blood-brain barrier and migrate along the olfactory neural route into the brain and cerebrospinal fluid. Our goal is to confirm this hypothesis by transnasally administrating Wharton’s Jelly mesenchymal stem cells (WJ-MSC) and neural progenitor cells (NPC) to perinatal rats in a model of hypoxic-ischemic brain injury. STUDY DESIGN: Four-day-old Wistar rat pups, previously brain-damaged by combined hypoxic-ischemic and inflammatory insult, either received WJ-MSC or green fluorescent protein-expressing NPC: The heads of the rat pups were immobilized and 3 ml drops containing the cells (50’000 cells/ml) were placed on one nostril allowing it to be snorted. This procedure was repeated twice, alternating right to left nostril with an interval of one minute between administrations. The rat pups received a total of 600’000 cells. Animals were sacrificed 24h, 48h or 7 days after the application of the cells. Fixed brains were collected, embedded in paraffin and sectioned. RESULTS: Transplanted cells were found in the layers of the olfactory bulb (OB), the cerebral cortex, thalamus and the hippocampus. The amount of cells was highest in the OB. Animals treated with transnasally delivered stem cells showed significantly decreased gliosis compared to untreated animals. CONCLUSION: Our data show that transnasal delivery of WJ-MSC and NPC to the newborn brain after perinatal brain damage is successful. The cells not only migrate the brain, but also decrease scar formation and improve neurogenesis. Therefore, the non-invasive intranasal delivery of stem cells to the brain may be the preferred method for stem cell treatment of perinatal brain damage and should be preferred in future clinical trials.

NonViral Gene Delivery of Growth and Differentiation Factor 6 (GDF6) to whole bovine Intervertebral Disc

Question: Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and de- generation. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM) [1]. Further studies showed that growth factors from the transforming growth factor (TGF) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC) [2]. Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods: Bovine IVD cells were isolated by pronase/collagenase II overnight digestion. After monolayer expansion up to passage 3, cells were transfected with the plasmid pGDF6 (RG211366, Origene, SF) or with green fluorescence protein (GFP) control using the NeonÒ transfection system (Invitrogen, Basel), both equipped with a Cy- tomegalovirus (CMV) promotor to induce over-expression. We tested a range of yet unpublished parameters for each of the primary disc cells to optimize efficiency. To test a non-viral gene therapy applied directly to 3D whole organ culture, bovine IVDs were harvested from fresh tails obtained from the abattoir within 5 h post-mortem [3]. Discs were then pre-incubated for 24 h in high glucose Dulbecco’s Modified Eagle Medium and 5 % fetal calf serum. Each disc was transfected by injection of 5 lg of plasmid GDF6 (Origene, RG211366) into the center by 25G needle and using Hamilton sy- ringe. Electroporation was performed using 2-needle array electrode or tweezertrodes; 8 pulses at 200mv/cm with an interval of 10 ms were applied using ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) (Fig. 1). After transfection discs were cultured for 72 h to allow expression of GFP or GDF6. Discs were then fixed, cryosectioned and analysed by immunofluorescence against GDF6. Results: We successfully transfected bovine NP and AF cells in monolayer culture with the two plasmids using a 1,400 V, 20 ms and 2 pulses with a *25 % efficiency using 0.15 M cells and 3 lg DNA (Fig. 1). Organ IVD culture transfection revealed GFP6 positive staining in the centre of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GFP posi- tive cells. Conclusions: We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgments: This study was supported by the Lindenhof Foundation ‘‘Forschung und Lehre’’ (Project no. 13-02-F). References 1. Roughly PJ (2004) Spine (Phila) 29:2691–2699 2. 3. Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014) Arthritis Res Ther 16:R67 Chan SC, Gantenbein-Ritter B (2012) J Vis Exp 60(60):e3490

Genetic and functional analyses of cell division proteins FtsZ and FtsA in Escherichia coli

In the current model for bacterial cell division, the FtsZ protein forms a ring that marks the division plane, creating a cytoskeletal framework for the subsequent action of other essential division proteins such as FtsA and ZipA. The putative protein complex ultimately generates the division septum. The essential cell division protein FtsZ is a functional and structural homolog of eukaryotic tubulin, and like tubulin, FtsZ hydrolyzes GTP and self-assembles into protein filaments in a strictly GTP-dependent manner. FtsA shares sequence similarity with members of the ATPase superfamily that include actin, but its actual function remains unknown. To test the division model and elucidate functions of the division proteins, this dissertation primarily focuses on the analysis of FtsZ and FtsA in Escherichia coli. ^ By tagging with green fluorescent protein, we first demonstrated that FtsA also exhibits a ring-like structure at the potential division site. The localization of FtsA was dependent on functional FtsZ, suggesting that FtsA is recruited to the septum by the FtsZ ring. In support of this idea, we showed that FtsA and FtsZ directly interact. Using a novel E. coli in situ assay, we found that the FtsA-FtsZ interaction appears to be species-specific, although an interspecies interaction could occur between FtsA and FtsZ proteins from two closely related organisms. In addition, mutagenesis of FtsA revealed that no single domain is solely responsible for its septal localization or interaction with FtsZ. To explore the function of FtsA, we purified FtsA protein and demonstrated that it has ATPase activity. Furthermore, purified FtsA stimulates disassembly of FtsZ polymers in a sedimentation assay but does not affect GTP hydrolysis of FtsZ. This result suggests that in the cell, FtsA may function similarly in regulating dynamic instability of the FtsZ ring during the cell division process. ^ To elucidate the structure-function relationship of FtsZ, we carried out thorough genetic and functional analyses of the mutagenized FtsZ derivatives. Our results indicate that the conserved N-terminal domain of FtsZ is necessary and sufficient for FtsZ self-assembly and localization. Moreover, we discovered a critical role for an extreme C-terminal domain of FtsZ that consists of only 12 residues. Truncated FtsZ derivatives lacking this domain, though able to polymerize and localize, are defective in ring formation in vivo as well as interaction with FtsA and ZipA. Alanine scanning mutagenesis of this region pinpointed at least five residues necessary for the function of FtsZ. Studies of protein levels and protein-protein interactions suggested that these residues may be involved in regulating protein stability and/or FtsZ-FtsA interactions. Interestingly, two of the point mutants exhibited dominant-negative phenotypes. ^ In summary, results from this thesis work have provided additional support for the division machinery model and will contribute to a better understanding of the coordinate functions of FtsA and FtsZ in the cell division process. ^

Functional genomics tools to decipher the pathogenicity mechanisms of the necrotrophic fungus Plectosphaerella cucumerina in Arabidopsis thaliana

The analysis of the interaction between Arabidopsis thaliana and adapted (PcBMM) and nonadapted (Pc2127) isolates of the necrotrophic fungus Plectosphaerella cucumerina has contributed to the identification of molecular mechanisms controlling plant resistance to necrotrophs.To characterize the pathogenicity bases of the virulence of necrotrophic fungi in Arabidopsis, we developed P. cucumerina functional genomics tools using Agrobacterium tumefaciens-mediated transformation.We generated PcBMM-GFP and Pc2127-GFP transformants constitutively expressing the green fluorescence protein (GFP), and a collection of random T-DNA insertional PcBMM transformants. Confocal microscopy analyses of the initial stages of PcBMM-GFP infection revealed that this pathogen, like other necrotrophic fungi, does not form an appressorium or penetrate into plant cells, but causes successive degradation of leaf cell layers

Functional identification and reconstitution of an odorant receptor in single olfactory neurons

The olfactory system is remarkable in its capacity to discriminate a wide range of odorants through a series of transduction events initiated in olfactory receptor neurons. Each olfactory neuron is expected to express only a single odorant receptor gene that belongs to the G protein coupled receptor family. The ligand–receptor interaction, however, has not been clearly characterized. This study demonstrates the functional identification of olfactory receptor(s) for specific odorant(s) from single olfactory neurons by a combination of Ca2+-imaging and reverse transcription–coupled PCR analysis. First, a candidate odorant receptor was cloned from a single tissue-printed olfactory neuron that displayed odorant-induced Ca2+ increase. Next, recombinant adenovirus-mediated expression of the isolated receptor gene was established in the olfactory epithelium by using green fluorescent protein as a marker. The infected neurons elicited external Ca2+ entry when exposed to the odorant that originally was used to identify the receptor gene. Experiments performed to determine ligand specificity revealed that the odorant receptor recognized specific structural motifs within odorant molecules. The odorant receptor-mediated signal transduction appears to be reconstituted by this two-step approach: the receptor screening for given odorant(s) from single neurons and the functional expression of the receptor via recombinant adenovirus. The present approach should enable us to examine not only ligand specificity of an odorant receptor but also receptor specificity and diversity for a particular odorant of interest.

Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human PEX1 cDNA (HsPEX1) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged “enhanced” green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family. A stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 → Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells. These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders.

Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum

A vestigial, nonphotosynthetic plastid has been identified recently in protozoan parasites of the phylum Apicomplexa. The apicomplexan plastid, or “apicoplast,” is indispensable, but the complete sequence of both the Plasmodium falciparum and Toxoplasma gondii apicoplast genomes has offered no clue as to what essential metabolic function(s) this organelle might perform in parasites. To investigate possible functions of the apicoplast, we sought to identify nuclear-encoded genes whose products are targeted to the apicoplast in Plasmodium and Toxoplasma. We describe here nuclear genes encoding ribosomal proteins S9 and L28 and the fatty acid biosynthetic enzymes acyl carrier protein (ACP), β-ketoacyl-ACP synthase III (FabH), and β-hydroxyacyl-ACP dehydratase (FabZ). These genes show high similarity to plastid homologues, and immunolocalization of S9 and ACP verifies that the proteins accumulate in the plastid. All the putatively apicoplast-targeted proteins bear N-terminal presequences consistent with plastid targeting, and the ACP presequence is shown to be sufficient to target a recombinant green fluorescent protein reporter to the apicoplast in transgenic T. gondii. Localization of ACP, and very probably FabH and FabZ, in the apicoplast implicates fatty acid biosynthesis as a likely function of the apicoplast. Moreover, inhibition of P. falciparum growth by thiolactomycin, an inhibitor of FabH, indicates a vital role for apicoplast fatty acid biosynthesis. Because the fatty acid biosynthesis genes identified here are of a plastid/bacterial type, and distinct from those of the equivalent pathway in animals, fatty acid biosynthesis is potentially an excellent target for therapeutics directed against malaria, toxoplasmosis, and other apicomplexan-mediated diseases.

A novel role for clathrin in cytokinesis

Using clathrin-minus Dictyostelium cells, we identified a novel requirement for clathrin during cytokinesis. In suspension culture, clathrin-minus cells failed to divide and became large and multinucleate. This cytokinesis deficiency was not attributable to a pleiotropic effect on the actomyosin cytoskeleton, since other cellular events driven by myosin II (e.g., cortical contraction and capping of concanavalin A receptors) remained intact in clathrin-minus cells. Examination of cells expressing myosin II tagged with green fluorescent protein showed that clathrin-minus cells failed to assemble myosin II into a functional contractile ring. This inability to localize myosin II to a particular location was specific for cytokinesis, since clathrin-minus cells moving across a substrate localized myosin II properly to their posterior cortexes. These results demonstrate that clathrin is essential for construction of a functional contractile ring during cell division.

Analysis of exocytosis mutants indicates close coupling between regulated secretion and transcription activation in Tetrahymena

Stimulation of regulated secretory cells promotes protein release via the fusion of cytoplasmic storage vesicles with the plasma membrane. In Tetrahymena thermophila, brief exposure to secretagogue results in synchronous fusion of the entire set of docked dense-core granules with the plasma membrane. We show that stimulation is followed by rapid new dense-core granule synthesis involving gene induction. Two genes encoding granule matrix proteins, GRL1 and GRL4, are shown to undergo induction following stimulation, resulting in ≈10-fold message accumulation within 1 h. The mechanism of induction involves transcriptional regulation, and the upstream region of GRL1 functions in vivo as an inducible promoter in a heterologous reporter construct using the gene encoding green fluorescent protein. Taking advantage of the characterized exocytosis (exo−) mutants available in this system, we asked whether the signals for regranulation were generated directly by the initial stimulation, or whether downstream events were required for transcription activation. Three mutants, with defects at three distinct stages in the regulated secretory pathway, failed to show induction of GRL1 and GRL4 after exposure to secretagogue. These results argue that regranulation depends upon signals generated by the final steps in exocytosis.